For several reasons, spacecraft are constructed in so-called clean rooms. Particles could affect the function of spacecraft instruments, and for missions under planetary protection limitations, the biological contamination has to be restricted as much as possible. The proper maintenance of clean rooms includes, for instance, constant control of humidity and temperature, air filtering, and cleaning (disinfection) of the surfaces. The combination of these conditions creates an artificial, extreme biotope for microbial survival specialists: spore formers, autotrophs, multi-resistant, facultative, or even strictly anaerobic microorganisms have been detected in clean room habitats. Based on a diversity study of European and South-American spacecraft assembly clean rooms, the European Space Agency (ESA) has initialized and funded the creation of a public library of microbial isolates. Isolates from three different European clean rooms, as well as from the final assembly and launch facility in Kourou (French Guiana), have been phylogenetically analyzed and were lyophilized for long-term storage at the German Culture Collection facilities in Brunswick, Germany (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen). The isolates were obtained by either following the standard protocol for the determination of bioburden on, and around, spacecraft or the use of alternative cultivation strategies. Currently, the database contains 298 bacterial strains. Fifty-nine strains are Gram-negative microorganisms, belonging to the α-, β- and γ-Proteobacteria. Representatives of the Gram-positive phyla Actinobacteria, Bacteroidetes/Chlorobi, and Firmicutes were subjected to the collection. Ninety-four isolates (21 different species) of the genus Bacillus were included in the ESA collection. This public collection of extremotolerant microbes, which are adapted to a complicated artificial biotope, provides a wonderful source for industry and research focused on very unusual properties of microbes. For ESA, this collection is an essential resource with which to evaluate the contamination potential of spacecraft-associated biology and validate new biological contamination control and reduction procedures.